Cocal vesiculovirus envelope pseudotyped retroviral vectors

ABSTRACT

Provided herein are Cocal  vesiculovirus  envelope pseudotyped retroviral vectors that exhibit high titers, broad species and cell-type tropism, and improved serum stability. Disclosed Cocal  vesiculovirus  envelope pseudotyped retroviral vectors may be suitably employed for gene therapy applications and, in particular, for the ex vivo and in vivo delivery of a gene of interest to a wide variety of target cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/175,376, filed May 4, 2009, and which provisionalpatent application is incorporated by reference in its entirety herein.

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

This disclosure was made, in part, in the course of research sponsoredby the National Institute of Health, grant numbers HL36444, DK47754,HL074162, DK07786, DK56465, HL53750, AI061839, and AI063959. The U.S.government has certain rights in this disclosure.

SEQUENCE LISTING

The present application includes a Sequence Listing in electronic formatas a txt file titled “Sequence_Listing 04May10,” which was created onMay 4, 2010 and which has a size of 21 kilobytes (KB). The contents oftxt file “Sequence_Listing 04May10” are incorporated by referenceherein.

BACKGROUND OF THE DISCLOSURE

1. Technical Field of the Disclosure

The present disclosure relates, generally, to the fields of virology,immunology, and molecular biology. More specifically, provided hereinare Cocal vesiculovirus envelope pseudotyped retroviral vectors andparticles that exhibit high titers, broad species and cell-type tropism,and improved serum stability. The presently disclosed Cocalvesiculovirus envelope pseudotyped retroviral vectors and particles maybe suitably employed for gene therapy applications and, in particular,for the ex vivo and in vivo delivery of a gene of interest to a widevariety of target cells.

2. Description of the Related Art

Retroviral vectors, in particular lentiviral vectors, have shown greatpromise for gene therapy in preclinical animal models and more recentlyalso in clinical studies. Lentiviral vectors have several advantagesover gammaretroviral vectors including the ability to more efficientlytransduce quiescent cells. Lewis et al., EMBO J. 11:3053-3058 (1992).This is thought to be due to their ability to enter the nucleusindependently of mitosis, but other differences may also be involved.Yamashita and Emerman, Virology 344:88-93 (2006). Lentiviral vectorsbased on human, simian, or feline immunodeficiency virus can transduce avariety of non-dividing target cells. Case et al., Proc. Natl. Acad.Sci. U.S.A. 96:2988-2993 (1999) and Reiser et al., Proc. Natl. Acad.Sci. U.S.A. 93:15266-15271 (1996). And, in contrast to gammaretroviralvectors, lentiviral vectors do not typically integrate very close topromoter regions. Schroder et al., Cell 110:521-529 (2002). This featureof lentiviral vectors is important for the safe application of genetherapy given the recent occurrences of leukemia in SCID-X1 patientstreated with gammaretroviral vectors. Hacein-Bey-Abina et al., Science302(5645):415-419 (2003) [erratum Science 302(5645):568 (2003)]. Inparticular, self-inactivating (SIN) lentiviral vectors are likely tohave an improved safety profile (Montini et al., Nat. Biotech.24:687-696 (2006)) and can be produced at high titer, which areimportant considerations when translating gene therapy approaches intothe clinic.

Lentiviral vectors and other retroviral vectors are most commonlypseudotyped with Vesicular Stomatitis Virus envelope glycoprotein(VSV-G), which confers a broad tropism and also allows for efficientconcentration by centrifugation. Burns et al., Proc. Natl. Acad. Sci.U.S.A. 90:8033-8037 (1993). VSV-G pseudotyped lentiviral vectors haveshown efficacy for gene transfer to a wide variety of tissues includingliver, muscle, brain, kidney, retina, and hematopoietic cells. Highlevels of gene transfer to hematopoietic repopulating cells have beenobtained in large animal models using VSV-G pseudotyped HIV-1 basedlentiviral vectors (Trobridge et al., Blood 111:5537-5543 (2008) andHorn et al., Blood 103:3710-3716 (2004)), and HIV-based lentiviralvectors are currently being used for clinical trials for hematopoieticstem cell (HSC) gene therapy.

There are, however, some disadvantages to using VSV-G to pseudotyperetroviral vectors. Toxicity is associated with the constitutiveexpression of VSV-G, which has made generation of stable packaging celllines difficult (Ory et al., Proc. Natl. Acad. Sci. U.S.A.93:11400-11406 (1996)) and vectors pseudotyped with the VSV-G envelopeglycoprotein are inactivated by human serum complement (DePolo et al.,Mol. Ther. 2:218-222 (2000)), which may limit the use of VSV-Gpseudotyped lentiviral vectors for gene therapy applications where thevector is delivered in vivo.

Alternate pseudotypes have been evaluated including the FelineEndogeneous Virus (RD114) glycoprotein which has shown promise for HSCgene transfer. Sandrin et al. developed an RD114-based envelopeglycoprotein with a modified transmembrane-region (RD114/TR) that allowsfor efficient pseudotyping of lentiviral vectors and also allows forefficient concentration of lentiviral vectors by centrifugation. Blood100:823-832 (2002). RD114/TR pseudotyped vectors mediate efficient genetransfer into human hematopoietic progenitors and NOD/SCID repopulatingcells (Di Nunzio et al., Hum. Gene Ther. 18:811-820 (2007)) and RD114pseudotyped vectors also mediate efficient gene transfer in large animalmodels. Neff et al., Mol. Ther. 9:157-159 (2004); Hu et al., Mol. Ther.8:611-617 (2003); and Kelly et al., Blood Cells, Molecules, & Diseases30:132-143 (2003). RD114 pseudotyped vectors are also resistant to humanserum complement (Cosset et al., J. Virol. 69:7430-7436 (1995) andSandrin et al., Blood 100:823-832 (2002)), and RD114 pseudotypedoncoretroviral vectors have been used for in vivo delivery in the canineX-SCID model. Ting-De Ravin et al., Blood 107:3091-3097 (2006). Thetiters reported for RD114/TR pseudotyped lentiviral vectors have,however, generally been lower than those obtained with VSV-G, which maylimit their utility for clinical gene therapy applications.

Cocal virus is in the Vesiculovirus genus, and is a causative agent ofvesicular stomatitis in mammals. Cocal virus was originally isolatedfrom mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242(1964)), and infections have been identified in Trinidad, Brazil, andArgentina from insects, cattle, and horses. Many of the vesiculovirusesthat infect mammals have been isolated from naturally infectedarthropods, suggesting that they are vector-borne. Antibodies tovesiculoviruses are common among people living in rural areas where theviruses are endemic and laboratory-acquired; infections in humansusually result in influenza-like symptoms. The Cocal virus envelopeglycoprotein shares 71.5% identity at the amino acid level with VSV-GIndiana, and phylogenetic comparison of the envelope gene ofvesiculoviruses shows that Cocal virus is serologically distinct from,but most closely related to, VSV-G Indiana strains among thevesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) andTravassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006(1984).

There remains an unmet need in the art for improved retroviral vectorsthat may be generated in sufficiently high titers to permit their use inex vivo and in vivo gene therapy applications, that exhibit broadspecies and cell-type tropism, and that are resistant to serumdegradation in vivo.

SUMMARY OF THE INVENTION

The present invention addresses these and other related needs byproviding, inter alia, Cocal vesiculovirus envelope pseudotypedretroviral vector particles that may be suitably employed for genetransfer applications including gene therapy and vaccines, and, inparticular, for the ex vivo and in vivo delivery of a gene of interestto a wide variety of target cells. The Cocal vesiculovirus envelopepseudotyped retroviral vector particles disclosed herein exhibit hightiters, broad species and cell-type tropism, and improved serumstability.

Thus, within certain embodiments, the present disclosure provides Cocalvesiculovirus envelope pseudotyped retroviral vector particlesincluding, for example, lentiviral, alpharetroviral, betaretroviral,gammaretroviral, deltaretroviral, and epsilonretroviral vector particlesthat comprise retroviral Gag, Pol, and/or one or more accessoryprotein(s) and a Cocal vesiculovirus envelope protein. Within certainaspects of these embodiments, the Gag, Pol, and accessory proteins arelentiviral and/or gammaretroviral. Within other aspects, the Cocalvesiculovirus envelope protein is encoded by a polynucleotide comprisingthe nucleotide sequence of SEQ ID NO: 1 and has the amino acid sequencepresented in SEQ ID NO: 2. Within yet other aspects, the Cocalvesiculovirus envelope protein is encoded by a polynucleotide comprisinga nucleotide sequence that is human codon-optimized such as, forexample, the nucleotide sequence of SEQ ID NO: 3. One such Cocalvesiculovirus envelope protein encoded by a human codon-optimizedpolynucleotide is exemplified by the amino acid sequence of SEQ ID NO:4.

Within other embodiments, the present disclosure providespolynucleotides comprising a human codon-optimized nucleotide sequenceencoding a Cocal vesiculovirus envelope protein. An exemplary humancodon-optimized nucleotide sequence is presented as SEQ ID NO: 3, whichsequence encodes the Cocal vesiculovirus envelope protein having theamino acid sequence of SEQ ID NO: 4.

Still further embodiments of the present disclosure provide plasmidvectors for the expression of a Cocal vesiculovirus envelope protein,which vectors comprise a polynucleotide encoding a Cocal vesiculovirusenvelope protein wherein the polynucleotide is under the transcriptionalcontrol of a eukaryotic transcriptional promoter. Exemplarypolynucleotides that encode a Cocal vesiculovirus envelope proteininclude the nucleotide sequence of SEQ ID NO: 1, which encodes theprotein having the amino acid sequence of SEQ ID NO: 2, as well as ahuman codon-optimized variant of that nucleotide sequence that ispresented as SEQ ID NO: 3 and which encodes the amino acid sequence ofSEQ ID NO: 4. An exemplary plasmid vector for the expression of a Cocalvesiculovirus envelope proteins is designated herein as pMD2.CocalG,which has the nucleotide sequence presented as SEQ ID NO: 5.

Other embodiments provide Cocal vesiculovirus envelope proteins, inparticular proteins encoded by human codon-optimized nucleotidesequences. An exemplary Cocal vesiculovirus envelope protein of thepresent disclosure has the amino acid sequence of SEQ ID NO: 4, whichmay be encoded by the nucleotide sequence of SEQ ID NO: 3.

Yet other embodiments of the present disclosure provide methods forgenerating Cocal envelope pseudotyped retroviral vector particles, whichmethods comprise the step of transfecting a cell with a retroviralvector plasmid, a retroviral helper plasmid or plasmids, and a plasmidvector for the expression of a Cocal vesiculovirus envelope protein.Exemplified herein are methods for generating Cocal envelope pseudotypedretroviral vector particles wherein the retroviral vector plasmids andretroviral helper plasmids are lentiviral vector plasmids and lentiviralhelper plasmids. Also exemplified herein are methods for generatingCocal envelope pseudotyped retroviral vector particles wherein theretroviral vector plasmids and retroviral helper plasmids aregammaretroviral vector plasmids and gammaretroviral helper plasmids. Itwill be understood, however, that other Cocal envelope pseudotypedretroviral vector particles including, for example, alpharetroviral,betaretroviral, deltaretroviral, and epsilonretroviral vector particlesmay also be generated by the presently disclosed methods by employingsuitable vector and helper plasmids that are known to those of skill inthe art.

Further embodiments of the present disclosure provide methods fordelivering a gene of interest to a target cell, which methods comprisethe step of contacting a target cell with a Cocal vesiculovirus envelopepseudotyped retroviral vector particle.

Still further embodiments of the present disclosure provide methods fordelivering a gene of interest into a hematopoietic system of a patient,which methods comprise the steps of: (a) harvesting CD34⁺ hematopoieticstem cells (HSCs) from the patient; (b) transducing the HSCs ex vivowith a Cocal vesiculovirus envelope pseudotyped retroviral vectorparticle comprising the gene of interest; and (c) introducing the exvivo transduced HSCs into the patient under conditions that allowreconstitution of the patient's hematopoietic system.

The present disclosure also provides methods for identifying a patientsusceptible to treatment with a Cocal vesiculovirus envelope pseudotypedretroviral vector particle, which methods comprise the steps of: (a)isolating a serum sample from the patient; (b) contacting the Cocalvesiculovirus envelope pseudotyped retroviral vector particle with theserum sample; and (c) testing the Cocal vesiculovirus envelopepseudotyped retroviral vector particle for serum inactivation.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All publications, patents, and patent applications citedherein, whether supra or infra, are hereby incorporated by reference intheir entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Envelope plasmids. All envelopes were expressed from a CMVpromoter with a human or rabbit beta globin intron (hBGint, rBGint) 5′to the ORF and a human or rabbit beta globin poly A sequence (hBGpA,rBGpA). The Cocal ORF was codon-optimized for human cells.

FIG. 2. Tropism of Cocal envelope relative to VSV-G and RD114/TRenvelopes. The indicated cell lines and primary cell cultures weretransduced at an MOI of 5 and the percentage of Enhanced GreenFluorescent Protein (EGFP)-expressing cells was determined 6 days aftervector exposure.

FIG. 3. Serum neutralization of lentiviral pseudotypes. Serum from 10humans (A), 5 dogs (B), and 5 macaques (C) were incubated with vector at37° C. for 30 minutes then added to HT1080 cells to evaluate the numberof EGFP-transducing units. EGFP expression was evaluated by flowcytometry for EGFP expression 3 days after vector exposure, and thepercentage of EGFP expressing cells after incubation in the serum wasdetermined relative to the percentage of EGFP expressing cells in thevector-only control to determine the fold increase or decrease in titerafter exposure to serum.

FIG. 4. Comparison of pseudotypes for gene transfer to hematopoieticprogenitors. CD34⁺ cells were exposed to vector at a MOI of 5 or 20 forhours then added to each well of a 24-well plate for CFU analysis. Thepercentage of EGFP-expressing cells was determined in liquid cultures 10days after vector exposure by flow cytometry and the percentage ofEGFP-expressing CFUs were determined by fluorescent microscopy 14 daysafter vector exposure.

FIG. 5. Engraftment and transgene expression levels in peripheral bloodcells of a pigtailed macaque that received both Cocal and VSV-Gpseudotyped lentivirally transduced stem cells. The percentages of EGFP(Cocal) and Enhanced Yellow Fluorescent Protein (EYFP; VSV-G)-expressingleukocytes in the peripheral blood detected by flow-cytometry are shown.

FIG. 6. Nucleotide sequence of Cocal vesiculovirus envelope glycoprotein(GenBank Accession No. AF045556; SEQ ID NO: 1).

FIG. 7. Amino acid sequence of Cocal vesiculovirus envelope glycoprotein(SEQ ID NO: 2) encoded by the nucleotide sequence of FIG. 6 (SEQ ID NO:1).

FIG. 8. Nucleotide sequence of the human codon enriched Cocalvesiculovirus envelope glycoprotein (SEQ ID NO: 3) based on thenucleotide sequence of FIG. 6 (SEQ ID NO: 1).

FIG. 9. Amino acid sequence of human codon enriched Cocal vesiculovirusenvelope glycoprotein (SEQ ID NO: 4) encoded by the nucleotide sequenceof FIG. 8 (SEQ ID NO: 3).

FIG. 10. Nucleotide sequence of the lentiviral vector designatedpMD2.CocalG (SEQ ID NO: 5).

FIG. 11. Pseudotyping gammaretroviral vectors. The titers of a Moloneyleukemia virus-based vector expressing EGFP by co-transfection of cocal,VSV, and RD114/TR envelope glycoproteins are compared.

FIG. 12. Efficient production of lentiviral vectors from 293C cells.

FIG. 13. Efficient marking in nonhuman primate long term hematopoieticrepopulating cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

As indicated above, the present disclosure is based on the discoverythat retroviral vectors may be efficiently pseudotyped using the Cocalvesiculovirus envelope glycoprotein described by Bhella et al., VirusRes. 54:197-205 (1998), and that Cocal vesiculovirus envelopeglycoprotein pseudotyped retroviral vectors can be produced at hightiter, are stable to ultracentrifugation and repeated freeze-thawcycles, exhibit broad species and cell-type tropism, can efficientlytransduce long-term repopulating cells in a clinically relevant nonhumanprimate model, are resistant to serum inactivation, and are lesssusceptible to in vivo immune responses in patients. Thus, the Cocalvesiculovirus envelope glycoprotein pseudotyped retroviral vectorsdisclosed herein will find broad utility in gene therapy applicationsincluding, for example, the transduction of hematopoietic progenitor orstem cells for the in vivo repopulation of a patient's hematopoieticcell system.

The present disclosure is exemplified by specific reference to Cocalpseudotyped lentiviral vectors and Cocal pseudotyped gammaretroviralvectors. It will be understood, that the teachings disclosed herein maybe extended with routine experimentation to the pseudotyping of otherviral vectors derived from other enveloped viruses, in particular viralvectors derived from the range of alternative retroviruses includingalpharetroviral, betaretroviral, deltaretroviral, and epsilonretroviral.The present disclosure further contemplates that the Cocal envelope maybe employed to pseudotype a wide variety of enveloped vectors that canbe pseudotyped with VSV-G such as, for example, VSV vectors, baculovirusvectors, and hybrid alphavirus/rhabdovirus vectors. Rose et al., Proc.Natl. Acad. Sci. USA. 105(15):5839-43 (2008); Rose et al., 8th ConfRetrovir. Oppor. Infect. 8:50 (2001); Cooper et al., J. Virol.82(1):207-19 (2008); Li et al., J. Gene Med. 11(2):150-9 (2009); and Liet al., J. Gene Med. 11(1):57-65 (2009). It is further contemplated thatthe Cocal envelope pseudotyped vectors disclosed herein may be superiorfor certain vaccine applications due to reduced serum neutralization.

VSV-G is the most commonly used envelope used to pseudotype lentiviralvectors due to its broad tropism and biophysical properties that allowfor generation of vector virions at high titer that are stable duringultracentrifugation and freeze-thawing. One drawback of VSV-Gpseudotyped lentiviral vectors, that is overcome by the presentlydisclosed Cocal envelope pseudotyped lentiviral vectors, is that humanserum can inactivate VSV-G pseudotyped virions thereby limiting theirutility for in vivo delivery. DePolo et al., Mol. Ther. 2:218-222 (2000)and Croyle et al., J. Virol. 78:912-921 (2004). Also, humans developpotent immune responses against VSV-G after the administration of VSV-Gvector transduced cells which can limit the efficacy of future infusionsof VSV-G pseudotyped vectors.

As disclosed herein, quite surprisingly, the Cocal vesiculovirusenvelope glycoprotein allows for efficient HIV-based lentiviralpseudotyping and gene transfer and confers stability duringultracentrifugation and multiple freeze-thaw cycles. Lentiviral vectorsmay be reproducibly generated and concentrated to over 10⁸ TU per ml.Cocal glycoprotein envelope exhibits a broad tropism and mediatesefficient transduction of human cell types from all tissues and speciestested. Cocal envelope is efficacious for gene delivery to severalimportant therapeutic target cell types including skin fibroblasts,stroma, and hematopoietic progenitors and can efficiently transduce aclinically relevant nonhuman primate competitive repopulation model.Lentiviral vectors pseudotyped with Cocal virus glycoprotein are lesssensitive to neutralization by human serum, as compared to lentiviralvectors pseudotyped with VSV and RD114/TR envelope glycoproteins, andexhibity utility for in vivo gene delivery applications.

In summary, the present disclosure provides a novel pseudotype forproduction of lentiviral vectors that can be efficiently concentrated tovery high titers and that also demonstrated efficient transduction ofseveral cell types important for gene therapy including primatehematopoietic repopulating cells. Cocal pseudotyped lentiviral vectorswill be useful for many therapeutic gene transfer applications and maybe particularly useful for in vivo delivery in humans due to theirresistance to inactivation by human serum.

Each of these embodiments of the present invention is described infurther detail herein below.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology andimmunology within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Fields et al., “Virology” (3^(rd)Edition, 1996); Sambrook, et al., “Molecular Cloning: A LaboratoryManual” (2^(nd) Edition, 1989); “DNA Cloning: A Practical Approach, vol.I & II” (D. Glover, ed.); “Oligonucleotide Synthesis” (N. Gait, ed.,1984); “Nucleic Acid Hybridization” (B. Hames & S. Higgins, eds., 1985);Perbal, “A Practical Guide to Molecular Cloning” (1984); Ausubel et al.,“Current Protocols in Molecular Biology” (New York, John Wiley and Sons,1987); Bonifacino et al., “Current Protocols in Cell Biology” (New York,John Wiley & Sons, 1999); Coligan et al., “Current Protocols inImmunology” (New York, John Wiley & Sons, 1999); Harlow and LaneAntibodies: a Laboratory Manual Cold Spring Harbor Laboratory (1988);and Lo, Ed., “Antibody Engineering: Methods and Protocols,” Part 1(Humana Press, Totowa, N.J., 2004). Techniques for producing both typesof mutations are well known in the art. For example, specific mutationscan be introduced using site-specific mutagenesis as described inSambrook et al., “Protocols in Molecular Biology,” supra. Randommutations in specific regions can be introduced using, for example,forced evolution as described in Gulick and Fahl, Proc. Natl. Acad. Sci.USA, 92:8140-8144 (1995).

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contextclearly dictates otherwise.

Cocal Vesiculovirus Envelope Pseudotyped Retroviral Vector Particles

As indicated above, the present disclosure is directed to Cocalvesiculovirus envelope pseudotyped retroviral vector particles,including, for example, lentiviral vector particles and gammaretroviralparticles, that exhibit high titers, broad species and cell-typetropism, and improved serum stability and, as a consequence, may besuitably employed for gene therapy applications and, in particular, forthe ex vivo and in vivo delivery of a gene of interest to a wide varietyof target cells.

The Cocal vesiculovirus envelope pseudotyped lentiviral vector particlesdisclosed herein comprise lentiviral Gag, Pol, and one or more accessoryprotein(s) and a Cocal vesiculovirus envelope protein. Exemplifiedherein are Cocal vesiculovirus envelope pseudotyped lentiviral vectorparticles wherein the envelope protein (SEQ ID NO: 2) is encoded by apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, aswell as envelope proteins encoded by human codon-optimized nucleotidesequences such as, for example, the nucleotide sequence of SEQ ID NO: 3.One such Cocal vesiculovirus envelope protein encoded by a humancodon-optimized polynucleotide is exemplified by the amino acid sequenceof SEQ ID NO: 4. Cocal envelope pseudotyped lentiviral vector particlesdescribed herein typically result in concentrated titers of at least10⁶, 10⁷, 10⁸, or 10⁹ transducing units (TU)/ml.

Species and cell-type tropism of the Cocal envelope pseudotypedlentiviral vector particles may be determined by transducing a widevariety of cell cultures such as, for example, Epstein-Barr virustransformed human B-lymphocytes, SV40-transformed fetus-derived humanretinal epithelia, SV40-transformed human iliac artery-derivedendothelial cells, untransformed Macaca nemestrina skin fibroblasts,untransformed Macaca mulatta skin fibroblasts, untransformed Macacafascicularis skin fibroblasts, untransformed Bos taurus skinfibroblasts, untransformed Canis familiaris skin fibroblasts,untransformed Felis catus skin fibroblasts, human IMR90 cells, D17canine osteosarcoma cells, 208F embryo fibroblasts, human Jurkat cells,K562 myelogenous leukemia cells, and macaque stromal cells.

Serum stability of the Cocal envelope pseudotyped lentiviral vectorparticles may be determined by mixing vector particle preparations withserum at 37° C. for 30 minutes followed by addition to HT1080 cells.Gene transfer was evaluated by flow cytometry for expression of a geneof interest 3 days after vector particle exposure, and determining thepercentage of cells expressing the gene of interest after incubation inthe serum relative to the percentage of cells expressing the gene ofinterest in a vector-only control to determine the fold increase ordecrease in titer after exposure to serum.

Plasmid Vectors Comprising a Polynucleotide Encoding a CocalVesiculovirus Envelope Protein

Still further embodiments of the present disclosure provide plasmidvectors for the expression of Cocal vesiculovirus envelope proteins,which vectors comprise a polynucleotide encoding a Cocal vesiculovirusenvelope protein wherein the polynucleotide is under the transcriptionalcontrol of a eukaryotic transcriptional promoter. Exemplarypolynucleotides that encode a Cocal vesiculovirus envelope proteininclude the nucleotide sequence of SEQ ID NO: 1 (GenBank Accession No.AF045556), which encodes the protein having the amino acid sequence ofSEQ ID NO: 2, as well as a human codon-optimized variant of thatnucleotide sequence that is presented as SEQ ID NO: 3 and which encodesthe amino acid sequence of SEQ ID NO: 4.

An exemplary plasmid vector for the expression of a Cocal vesiculovirusenvelope proteins is designated herein as pMD2.CocalG, which has thenucleotide sequence presented as SEQ ID NO: 5. The pMD2.CocalG plasmidis derived from the pMD2.G described by Didier Trono and that isavailable from Addgene (Cambridge, Mass., Plasmid No. 12259). Morespecifically, the pMD2.CocalG plasmid was generated by removing theVSV-G coding sequence from the pMD2.G plasmid and ligating apolynucleotide encoding a Cocal vesiculovirus envelope protein betweenthe human β-globin intron and polyadenylation sequences and downstreamof the constitutively active CMV promoter. In the case of thepMD2.CocalG plasmid described herein, the polynucleotide encoding theCocal vesiculovirus envelope protein was first human codon-optimizedusing GeneMaker technology (Blue Heron Biotechnology, Bothell, Wash.).It will be understood that this exemplary polynucleotide (SEQ ID NO: 3)is representative of a wide range of human codon-optimizedpolynucleotides that may be employed in the presently disclosed plasmidvectors for the expression of a Cocal vesiculovirus envelope protein.

Cocal Vesiculovirus Envelope Proteins

Cocal vesiculovirus envelope proteins have been described in theliterature (see, for example, Bhella et al., Virus Res. 54:197-205(1998) and GenBank Accession No. AF045556). The present disclosure alsoprovides Cocal vesiculovirus envelope proteins encoded by humancodon-optimized nucleotide sequences. An exemplary Cocal vesiculovirusenvelope protein of the present disclosure has the amino acid sequenceof SEQ ID NO: 4, which may be encoded by the human codon-optimizednucleotide sequence of SEQ ID NO: 3.

Methods for Generating Cocal Envelope Pseudotyped Retroviral VectorParticles

The present disclosure provide methods for generating Cocal envelopepseudotyped retroviral vector particles that comprise the step oftransfecting a cell with a retroviral vector plasmid, a retroviralhelper plasmid, and a plasmid vector for the expression of a Cocalvesiculovirus envelope protein. Exemplified herein are methods forgenerating Cocal envelope pseudotyped lentiviral vector particles thatcomprise the step of transfecting a cell with a lentiviral vectorplasmid, a lentiviral helper plasmid, and a plasmid vector for theexpression of a Cocal vesiculovirus envelope protein. It will beunderstood by those of skill in the art that these methods may beapplied, with routine experimentation, to the generation of Cocalenvelope pseudotyped retroviral vector particles includingalpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, andepsilonretroviral vector particles.

A vide variety of methodologies are available in the art for thegeneration of envelope pseudotyped lentiviral vector particles that maybe modified by employing a plasmid vector for the expression of a Cocalvesiculovirus envelope protein, as described herein, to achieve Cocalenvelope pseudotyped lentiviral vector particles. For example, suchlentiviral vector particles may be produced by transient transfection ofhuman cells, such as embryonic kidney (HEK) 293 cells by the calciumphosphate method or by the polyethylenimine (PEI) method with acombination of a lentiviral vector plasmid; a lentiviral helper plasmid;and a Cocal vesiculovirus envelope protein plasmid.

Suitable lentiviral vector plasmids include, for example, the SIN HIVvector plasmids pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene Plasmid No. 12252)and pRRLSIN.cPPT.PGK-YFP.WPRE (Naldini, San Raffaele Telethon Institutefor Gene Therapy, Italy), both of which contain a central polypurinetract, a woodchuck post-transcriptional regulatory element, and aninternal phosphoglycerate kinase (PGK) promoter driving expression ofenhanced green fluorescent protein (EGFP) or enhanced yellow fluorescentprotein (EYFP). An exemplary helper plasmid is pCMVΔR8.74 described byDull et al., J. Virol. 72:8463-8471 (1998).

Methods Employing Cocal Envelope Pseudotyped Lentiviral Vector Particles

The present disclosure provide methods for delivering a gene of interestto a target cell that comprise the step of contacting a target cell witha Cocal vesiculovirus envelope pseudotyped lentiviral vector particleand entry of the particle.

The present disclosure exemplifies these methods by describing thedelivery of a gene of interest into a hematopoietic system of a patient,which methods comprise the steps of: (a) harvesting CD34⁺ hematopoieticstem cells (HSCs) from the patient; (b) transducing the HSCs ex vivowith a Cocal vesiculovirus envelope pseudotyped lentiviral vectorparticle comprising the gene of interest; and (c) introducing the exvivo transduced HSCs into the patient under conditions that allowreconstitution of the patient's hematopoietic system.

Hematopoietic stem cells may be harvested from a patient, transduced exvivo with Cocal vesiculovirus envelope pseudotyped lentiviral vectorparticles, then reintroduced to reconstitute the entire hematopoieticsystem. Human CD34⁺ cells may be isolated from a patient by harvestingmarrow cells, labeling with anti-CD34+ monoclonal antibodies, andseparating the CD34+ cells by magnetic beads, such as MACS IgMmicrobeads (Miltenyi Biotec, Auburn, Calif.) according to themanufacturer's instructions. Trobridge et al., Blood 111:5537-5543(2008). Human CD34⁺ cells may be cultured in IMDM with FBS, Flt-3ligand, stem cell factor, thrombopoietin, interleukin 3, interleukin 6,and granulocyte colony-stimulating factor).

CD34+ cells are typically transduced by pretreatment with CH-296fibronectin fragment (Takara, New York, N.Y.) and exposed to Cocalvesiculovirus envelope pseudotyped lentiviral vector particles a MOI of5 for 20 hours. Cells may then be washed, resuspended in medium, andtested for transduction by, for example, CFU analysis or flow cytometryor by PCR analysis. After exposure to vector preparations, the HSCs maybe reintroduced into the patient to allow reconstitution of thepatient's hematopoietic system.

The present disclosure further provides methods for identifying apatient susceptible to treatment with a Cocal vesiculovirus envelopepseudotyped lentiviral vector particle that comprise the steps of: (a)isolating a serum sample from the patient; (b) contacting the Cocalvesiculovirus envelope pseudotyped lentiviral vector particle with theserum sample; and (c) testing the Cocal vesiculovirus envelopepseudotyped lentiviral vector particle for serum inactivation.

The following Examples are offered by way of illustration, notlimitation. The following non-limiting examples are provided toillustrate various aspects of the present disclosure. All references,patents, patent applications, published patent applications, and thelike are incorporated by reference in their entireties herein.

EXAMPLES Example 1 Materials and Methods

Construction of a Cocal Envelope Pseudotype Plasmid

The deduced open reading frame (ORF) from the published sequence ofCocal envelope (Bhella et al., Virus Res. 54:197-205 (1998); GenBankAccession No. AF045556; SEQ ID NO: 1) was used to generate a humancodon-optimized polynucleotide (SEQ ID NO: 3) that was synthesized byBlue Heron Biotechnology (Bothell, Wash.) using GeneMaker technologyaccording to the sequence specified with ClaI and StuI flankingrestriction sites. The optimized Cocal ORF was subcloned by standardtechniques into pMD2.G kindly provided by Didier Trono, Lausanne,Switzerland (Addgene Plasmid No. 12259) replacing the VSV-G ORF tocreate pMD2.CocalG.

Production of Pseudotyped Lentiviral Vector Preparations andDetermination of Titer

The SIN HIV vector plasmids used were pRRLSIN.cPPT.PGK-GFP.WPRE (AddgenePlasmid No. 12252) and pRRLSIN.cPPT.PGK-YFP.WPRE (kindly provided byLuigi Naldini, San Raffaele Telethon Institute for Gene Therapy, Italy),which contain a central polypurine tract, a woodchuckpost-transcriptional regulatory element, and an internalphosphoglycerate kinase (PGK) promoter driving expression of enhancedgreen fluorescent protein (EGFP) or enhanced yellow fluorescent protein(EYFP).

HIV-based vector particles were produced by transient transfection ofhuman embryonic kidney (HEK) 293 cells (Graham et al., J. Gen. Virol.36:59-74 (1977)) by calcium phosphate method as previously described(Horn et al., Blood 103:3710-3716 (2004)) or by polyethylenimine (PEI)method (Boussif et al., Proc. Natl. Acad. Sci. U.S.A. 92:7297-7301(1995)) with 27 μg lentiviral vector plasmid; 17.5 μg lentiviral helperplasmid pCMVΔR8.74 (Dull et al., J. Virol. 72:8463-8471 (1998)); andenvelope plasmid (3 μg Cocal, 6 μg VSV-G, or 9 μg RD114/TR)). Forpolyethylenimine-mediated transfection the plasmid DNA was added to 2 mlof serum-free DMEM and 3 μL of 1 μg/μL PEI (25 kDa Linear Catalog No.23966 from Polysciences, Inc., Warrington, Pa.) per μg of DNA was addedand the solution was immediately mixed by vortexing for 10 seconds andallowed to stand at room temperature for 15 minutes. The solution wasthen added dropwise to the HEK 293 cells and all subsequent steps werethe same as for calcium phosphate-mediated transfection.

Vector particles were concentrated 100-fold by centrifugation for 20-22hours at 6300 g at 4° C. For freeze-thaw experiments, the vectorparticles were frozen at −70° C. for 1 hour then placed in a 37° C.water bath until completely thawed and then removed to room temperature.The titer of vector particle preparations was determined by addingvector particle preparations to HEK 293 cells or human HT1080fibrosarcoma cells (Rasheed et al., Cancer 33:1027-1033 (1974)) platedat 1×10⁵ cells/ml the day before vector particle addition. Protaminesulfate was added immediately before addition of vector particle at afinal concentration of 8 μg/ml. Transduced cells were assayed by flowcytometry 3-4 days after vector exposure, and the percentage ofEGFP-expressing cells was used to calculate the number of EGFPtransducing units (TU)/ml of vector preparation.

Evaluation of Cocal Tropism in Cell Lines and Primary Cell Cultures

All cell cultures were supplemented with 10% fetal bovine serum (FBS)and penicillin and streptomycin unless otherwise stated. Epstein-Barrvirus transformed human B-lymphocytes (Cat. No. AG09387),SV40-transformed fetus-derived human retinal epithelia (Cat. No.AG06096), SV40-transformed human iliac artery-derived endothelial cells(Cat. No. AG10427), untransformed Macaca nemestrina skin fibroblasts(Cat. No. AG08426), untransformed Macaca mulatta skin fibroblasts (Cat.No. AG06249), untransformed Macaca fascicularis skin fibroblasts (Cat.No. AG21329), untransformed Bos taurus skin fibroblasts (Cat. No.AG08130), untransformed Canis familiaris skin fibroblasts, anduntransformed Felis catus skin fibroblasts (Cat. No. GM06207) wereobtained from the Coriell Institute for Medical Research (Camden, N.J.and cultured as directed. Human IMR90 cells were obtained from theAmerican Type Culture Collection (ATCC Cat. No. CCL-186) and cultured inEagle's Minimum Essential Medium with 15% FBS. D17 canine osteosarcomacells (ATCC CRL-6248) and rat 208F embryo fibroblasts (Quade, Virol.98:461-465 (1979)) were cultured in Dulbecco's Modified Eagle Medium(DMEM). Human Jurkat cells were cultured in RPMI-1640. Gillis andWatson, J. Exp. Med. 152:1709-1719 (1980). K562 myelogenous leukemiacells (Lozzio and Lozzio, Blood 45(3):321-334 (1975)) were cultured inIscove's Modified Dulbecco's Medium (IMDM). Macaque stromal cells wereisolated from Macaca nemestrina bone marrow aspirates that were depletedof red blood cells by two washes with hemolytic buffer (150 mM ammoniumchloride, 12 mM sodium bicarbonate, 0.1 mM EDTA), and nucleated cellswere plated at 1×10⁶ to 2×10⁶ cells/ml in tissue culture-treated T-75flasks and cultured for 3 to 4 weeks in alpha minimum essential mediumwith 20% FBS and L-glutamine.

For the first 3 days after plating, one-half of the media was replacedin each flask daily, and after the first 3 days, the medium was changedevery third day. Cells were passaged once or twice per week uponreaching approximately 80% confluence. The absence of hematopoieticmarkers was confirmed after 3 to 4 weeks of culture by immunostainingand flow cytometry. For all cell types, cells were plated at 1×10⁵cells/ml the day before vector addition and vector was added at an MOIof 5 based on the titer on HEK 293 cells. Gene transfer was evaluated byflow cytometry for EGFP expression 6 days after vector exposure.

Serum Neutralization Assays

Human serum samples were obtained under an institutional review board(IRB)-approved protocol at Fred Hutchinson Cancer Research Center andstored frozen at −20° C. prior to use. Dogs were raised and housed atthe Fred Hutchinson Cancer Research Center (FHCRC) and macaques werehoused at the University of Washington National Primate Research Centerunder conditions approved by the American Association for Accreditationof Laboratory Animal Care. Twenty μl of serum were mixed with VSV-G,Cocal, or RD114/TR vector preparations with 5×10⁵ (Cocal and VSV-G) or5×10⁴ (RD114/TR) EGFP TU in triplicate and incubated at 37° C. for 30minutes then added to 1×10⁵ HT1080 cells. A vector-only control was alsoincubated at 37° C. for 30 min. Gene transfer was evaluated by flowcytometry for EGFP expression 3 days after vector exposure, and thepercentage of EGFP-expressing cells after incubation in the serum wasdetermined relative to the percentage of EGFP-expressing cells in thevector-only control to determine the fold increase or decrease in titerafter exposure to serum.

Isolation and Transduction of CD34⁺ Hematopoietic Progenitors

Human CD34⁺ cells were collected from a volunteer under an institutionalreview board approved protocol and isolated by magnetic beads (MiltenyiBiotec, Auburn, Calif.) according to the manufacturer's instructions andstored in liquid nitrogen until use. Bone marrow CD34-enriched primatecells were isolated using the 12.8 IgM anti-CD34⁺ antibody and MACS IgMmicrobeads (Miltenyi Biotec, Auburn, Calif., United States) according tothe manufacturer's instructions as previously described. Trobridge etal., Blood 111:5537-5543 (2008). Canine bone marrow CD34⁺ were isolatedas previously described (Goerner et al., Blood 98:2065-2070 (2001) andNeff et al., Blood 100:2026-2031 (2002)) using the biotinylatedmonoclonal antibody 1H6 (IgG1 anti-canine CD34) and used withoutcryopreservation.

Human CD34⁺ cells were thawed and cultured overnight in IMDM with 10%FBS, 100 ng/ml Flt-3 ligand, 100 ng/ml stem cell factor [SCF], 100 ng/mlthrombopoietin [TPO], 100 ng/ml interleukin 3 [IL-3], 100 ng/mlinterleukin 6 [IL-6] and 100 ng/ml granulocyte colony-stimulating factor[G-CSF]). 1×10⁵ CD34⁺ cells were then added to each well of a 24-wellplate pretreated with 50 μg/ml CH-296 fibronectin fragment (Takara, NewYork, N.Y.) and exposed to vector at a MOI of 5 for 20 hours with 4μg/mL protamine sulfate. Cells were then washed, resuspended in medium,and plated in tissue culture treated 24-well plates or plated for CFUanalysis or cultured for 10 days and analyzed by flow cytometry on day10. For CFU assay CD34-enriched cells (3000 per 35-mm plate) werecultured in a double-layer agar culture system. Briefly, isolated cellswere cultured in MEM alpha medium (Invitrogen, Carlsbad, Calif.)supplemented with 25% FBS (Hyclone, Logan, Utah), 0.1% BSA (fraction V;Sigma), 1 mM L-Glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin and0.3% (wt/vol) SeaPlaque agarose (Cambrex, East Rutherford, N.J.)overlaid on MEM alpha medium with 0.5% SeaPlaque agarose (wt/vol)containing 100 ng/mL of SCF, MGDF, IL-3, IL-6, granulocyte-macrophagecolony-stimulating factor (GM-CSF), G-CSF, 4 U/mL erythropoietin, 25%FBS, 0.1% BSA, 1 mM L-Glutamine, 50 U/mL penicillin and 50 μg/mLstreptomycin. Cultures were incubated at 37° C. in 5% CO2 in ahumidified incubator. All CFU cultures were performed in triplicate. Thetotal number, as well as the number of EGFP-positive colonies, wasenumerated at day 14 of culture by fluorescence microscopy to determinethe percentage of cells expressing the transgene.

CD34-enriched primate cells were thawed and cultured in IMDM with 10%FBS with 100 U/mL penicillin and 100 μg/mL streptomycin, supplementedwith 100 ng/mL rhSCF, 100 ng/mL rhuFlt3-L, 100 ng/mL interleukin (IL)-3,100 ng/mL IL-6, 100 ng/mL TPO, and 100 ng/mL G-CSF for 15-18 hoursbefore transduction. For transduction, cells were supplemented with thesame cytokine combination in flasks previously coated with the CH-296fragment of fibronectin (Retronectin, Takara, Shiga, Japan) and with 4μg/mL protamine sulfate. The cells were exposed to an initial dose ofvector for 16 hours and then exposed to second dose of vector overnightfor 8 hours, then washed, resuspended in medium and plated in 24-wellplates. The cells were analyzed by flow cytometry for EGFP expression onday 10.

Canine CD34-enriched cells were cultured in IMDM with 10% FBS, 10 U/mLpenicillin and 100 μg/mL streptomycin, supplemented with 50 ng/mL canineG-CSF, 50 ng/mL canine SCF, 50 ng/mL rhFlt3-L, and 50 ng/mL rhMGDF.1×10⁵ freshly isolated CD34⁺ cells were plated in each well of a 12-wellplate pretreated with 50 μg/ml CH-296 fibronectin fragment (Takara, NewYork, N.Y.) and supplemented with 4 μg/mL protamine sulfate. The cellswere exposed to vector in triplicate at an MOI of 5 for 20 hours, thenwashed, resuspended in medium and plated in 24-well plates and analyzedby flow cytometry on day 10.

Analysis of Marking in the Macaque Model Using a CompetitiveRepopulation Assay

Macaque CD34⁺ cells were isolated and transductions were performed asindicated above except that cells were exposed to an initial dose ofvector for 6.5-8 hours and then exposed to a second dose of vectorovernight for 17-18 hours at an MOI of 20. Following transduction, cellswere infused into the recipient after myeloablative total bodyirradiation (TBI) conditioning. Animal L06348 received a myeloablativedose of 1020 cGy TBI in four doses of 255 cGy, from a single sourcelinear X-ray accelerator (Linac Systems, Inc., Lakewood, N.J., USA) at 7cGy/minute. After infusion of autologous gene-modified cells, theanimals received recombinant G-CSF at 100 μg/kg daily until the animalsmaintained an ANC of >500/μL. To suppress a potential immune response tothe EGFP and EYFP proteins, tacrolimus was orally administered daily ata concentration of 2.5 mg/kg 2 days prior to infusion of vector-exposedcells and continued for 10 days. The concentration was then increased to3.5 mg/kg until 56 days post-transplant then tapered to 1.2 mg/kg untilday 65 post-transplant. The animal also received standard supportivecare including intravenous hydration and broad spectrum antibiotics(cetazadime, vancomycin, gentamicin), an antiviral agent (acyclovir), anantifungal agent (fluconazole) and transfusions with irradiatedpigtailed macaque whole blood for treatment of post-transplantthrombocytopenia. Leukocytes were isolated by ammonium chloride red celllysis from heparinized peripheral blood and bone marrow samples drawn atmultiple time points after transplantation were analyzed for EGFPexpression on a FACSVantage or FACSCalibur (Becton-Dickinson, San Jose,Calif.). Transgene expression in granulocyte, monocyte, and lymphocytepopulations was determined by gating based on either forward andright-angle light scatter characteristics or expression oflineage-specific CD markers. The antibodies used for lineage-specificmarkers included CD3 (clone SP34-2), CD13 (clone L138), CD20 (cloneL27), and CD34 (clone 563). All antibodies were supplied by BectonDickinson (Franklin Lakes, N.J.) and conjugated to phycoerythrin. Redcells and platelets from whole blood diluted in phosphate-bufferedsaline were delineated by forward and right-angle light scatterproperties and assessed for EGFP or EYFP expression.

Example 2 Construction of a Cocal Expression Plasmid and Pseudotyping ofLentiviral Vectors

A human codon optimized version of Cocal DNA was synthesized based onthe amino acid sequence reported by Bhella et al., Virus Research54:197-205 (1998). The optimized sequence was cloned into the plasmidbackbone that expresses the Cocal envelope glycoprotein from a CMVpromoter with a human β-globin intron and polyadenylation sequence (FIG.1). This plasmid backbone is identical to the plasmid backbone forpMD.2G, a VSV-G plasmid routinely used for the production of VSV-Gpseudotyped lentiviral vectors.

SIN lentiviral vectors that express the enhanced green fluorescent(EGFP) protein from a phosphoglycerate kinase (PGK) promoter weregenerated by transient transfection and pseudotyped with Cocal, VSV-G,or RD114/TR envelope. Viral titers were determined on HEK 293 cells andalso on human HT1080 fibrosarcoma cells. Protamine sulfate and polybreneare cationic polymers commonly used to enhance infection withretroviruses (Manning et al., Appl. Microbiol. 22:1162-1163 (1971)) andtransduction with retroviral vectors (Cornetta and Anderson, J. Virol.Methods 23:187-194 (1989)) including vectors pseudotyped with VSV-G andRD114/TR. Transduction with and without protamine sulfate were comparedusing Cocal pseudotyped lentiviral vectors. The addition of protaminesulfate at a concentration of 8 μg/ml enhanced transduction of HT1080cells approximately seven-fold, so protamine sulfate was included duringvector exposure for all subsequent experiments.

When producing VSV-G pseudotyped lentiviral vectors by transienttransfection, varying the amount of envelope plasmid can significantlyaffect titers. Lentiviral vectors were, therefore, prepared that expressthe enhanced green fluorescent (EGFP) protein with varying amounts ofeach envelope pseudotype and titers were compared on HEK 293 cells. Forall three pseudotypes, titers varied with the amount of envelopeglycoprotein used in the transient transfection; titers also variedslightly between different plasmid preparations. High titers wereroutinely achieved using either 3 μg of Cocal, 6 μg of VSV-G, or 9 μg ofthe RD114/TR envelope plasmid in a standard transient transfectionprotocol which also included 27 μg of vector plasmid and 17.5 μg ofhelper plasmid to transfect 1.2×10⁷ HEK 293 cells. The Cocal envelopeplasmid had a human codon-optimized ORF which increased the efficiencyof Cocal envelope glycoprotein production in transfected HEK 293 cells.

Vector stocks were prepared in triplicate, concentrated 100-fold bycentrifugation, and the titers were compared before and after a singlefreeze thaw or after 3 freeze-thaw cycles (Table 1).

TABLE 1 Efficiency of Concentration and Freeze-thaw Stability ofLentiviral Pseudotypes 1X Freeze- 3X Freeze- Concentrated Concentrationthaw thaw Pseudotype Titer Titer (100X) efficiency* efficiency^(†)efficiency^(†) Cocal 5.7 × 10⁶ ± 3.3 × 10⁵ 3.0 × 10⁸ ± 1.7 × 10⁷   53% ±3.9% 94.7% ± 4.1% 83.5% ± 2.2% VSV-G 5.8 × 10⁶ ± 9.2 × 10⁴ 3.7 × 10⁸ ±2.0 × 10⁷ 63.1% ± 2.9% 85.9% ± 2.6% 79.3% ± 4.0% RD114/TR 5.2 × 10⁴ ±1.9 × 10³ 1.3 × 10⁶ ± 2.1 × 10⁵ 24.8% ± 3.1%  88.7% ± 46.7%  62.8% ±16.2% *The percent of EGFP transducing units remaining after 100-foldconcentration. ^(†)The percent of EGFP transducing units remaining afterthe indicated number of freeze-thaw cycles.Lentiviral vectors prepared with either the Cocal or the VSV-G envelopereproducibly resulted in concentrated titers of approximately 10⁸ EGFPtransducing units (TU)/ml, while pseudotyping with the RD114/TR envelopeyielded lower titers and was more variable with ranges between 10⁶ and10⁷ TU/ml. The Cocal envelope allowed for efficient concentration bycentrifugation and efficiently retained titer at similar levels to VSV-Gduring multiple freeze-thaw cycles.

Example 3 Cocal Pseudotyped Lentiviral Vectors have a Broad Tropism

The high titers of Cocal pseudotyped vector preparations suggested thatthey will be highly effective for many gene transfer and gene therapyapplications. The efficiency of transduction was compared for a panel ofcell lines and primary cells derived from several tissues that areimportant targets for gene therapy, and from several species commonlyused as preclinical models for gene therapy. FIG. 3 shows the relativetransduction efficiency of transformed human cell lines and primarycells derived from blood, retinal epithelia, lung fibroblasts, boneendothelia, skin fibroblasts, stroma from humans, three species ofmacaques, cow, dog, cat or rat. The Cocal envelope allowed for highlyefficient transduction of cell types from all tissues and most speciestested with the exception of rhesus (Macaca mulatta) and cynomolgous orcrab-eating macaque (Macaca fascicularis) nonhuman primate cells wherethe transduction rates were very low for all pseudotypes. This was asexpected due to host cell restriction of the HIV-based lentiviral vectortransduction by Trim 5α. Stremlau et al., Nature 427:848-853 (2004). Inpigtailed macaque (Macaca nemestrina) cells, defective Trim5α isoforms(Brennan et al., J. Virol. 81(22):12210-7 (2007)) allows for infectionwith HIV-1 (Agy et al., Science 257:103-106 (1992)) and also forefficient gene transfer with lentiviral vectors. Trobridge et al., Blood111:5537-5543 (2008).

Overall, gene transfer was similar for Cocal and VSV-G in all tissuesand species tested with the exception of macaques, where Cocal envelopemediated higher gene transfer efficiency relative to VSV-G in all fiveprimary cell types tested. The transduction efficiency using Cocalpseudotyped vectors was also similar to RD114/TR pseudotyped vectorexcept in the cynomolgous or crab-eating macaque (Macaca fascicularis),where RD114/TR mediated the most efficient gene transfer, and also incat and rat fibroblasts where both Cocal and VSV-G pseudotyped vectorsmediated much higher transduction efficiencies than the RD114/TRpseudotype.

Example 4 Cocal Pseudotyped Lentiviral Vectors are Resistant to HumanSerum

The high titer, broad tropism, and stability of Cocal pseudotypedlentiviral vectors suggested that will also be highly effective for invivo delivery of transgenes. One limitation of in vivo gene deliveryusing VSV-G pseudotyped lentiviral vectors is that in humans serumneutralization of VSV-G pseudotyped vectors limits their effectiveness.DePolo et al., Mol. Ther. 2:218-222 (2000). VSV-G and Cocalvesiculovirus envelope glycoproteins have 71.5% identity at the aminoacid level, and VSV-G and Cocal vesiculoviruses are distinctserologically. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) andTravassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006(1984).

The ability of serum from 10 individuals to inactivate lentiviral vectorvirions produced using each pseudotype. The relative sensitivity toserum neutralization of VSV-G, Cocal, and RD114/TR pseudotypedlentiviral vectors was also compared in canines and macaques, twocommonly used large animal models for preclinical gene therapy studies.Vector preparations were incubated with or without serum at 37° C.before being plated onto cells, and the number of EGFP-transducing unitsremaining were determined relative to the vector-only control (FIG. 3).For Cocal and VSV-G pseudotypes the level of serum inactivation variedsignificantly between human individuals (p<0.0001). The level of serumresistance was not significantly different between individuals forRD114/TR (p=0.12). In humans, Cocal pseudotyped vector virions wereoverall relatively resistant to serum neutralization. In 7/10individuals VSV-G pseudotyped vectors were neutralized significantlymore than Cocal pseudotyped vectors (Pts. #2, 3, 5, 6, 9, p<0.01, Pts#7, 10 p<0.05). The RD114/TR pseudotyped vector was inactivated morethan Cocal pseudotyped vector in 6/10 individuals (Pts. #4, 5, 6, 7, 9,10). One individual's (Pt. #8) serum had a higher neutralizing activityto Cocal than VSV-G or RD114/TR but the difference was not significant(p=0.95 and p=0.31 respectively). Overall, these data suggest that Cocalpseudotyped vectors may be more effective than VSV-G or RD114/TRpseudotyped vectors for in vivo delivery in humans, since they are moreresistant to inactivation when incubated in human serum. The ability toproduce Cocal pseudotyped vectors at approximately 100-fold highertiters than RD114/TR pseudotypes (Table 1) should also be taken intoconsideration for in vivo applications.

The relative sensitivity to serum neutralization of VSV-G, Cocal andRD114/TR pseudotyped lentiviral vectors was also compared in dogs, acommonly used large animal model for preclinical gene therapy studies.For all pseudotypes, the level of inactivation varied significantlybetween dogs (p<0.001 for RD114/TR and VSV-G and p<0.05 for Cocal).Canine serum potently inactivated both Cocal and VSV-G pseudotypedvectors, and RD114/TR was significantly more resistant than either Cocalor VSV-G (p<0.01) to inactivation in all 5 dogs (FIG. 3B).

We also compared neutralization of VSV-G and Cocal envelope pseudotypedvectors in five macaques (FIG. 3C). The level of inactivation variedsignificantly between animals for Cocal (p<0.05), but not for VSV-G(p=0.95) pseudotyped vectors. In 4/5 animals there was no significantdifference in the level of inactivation for Cocal or VSV-G pseudotypes,and in one monkey VSV-G pseudotyped lentiviral vectors were moreresistant to neutralization than Cocal pseudotyped vectors.

The Cocal envelope pseudotyped vector virions disclosed herein areresistant to inactivation when incubated with human serum in 70% of thepatients tested. This suggests that Cocal pseudotyped vectors may bemore effective for in vivo gene delivery in individuals having highlevels of pre-existing serum neutralizing activity to VSV-G.Additionally, the unexpected observation that different individualsexhibit different pre-existing serum resistance to Cocal and VSV-Gpseudotypes suggests that the selection of pseudotype for in vivodelivery can be tailored for a specific individual by performing asimple serum neutralization test prior to vector administration.

In canine and macaque serum, significant differences were observed inneutralization for Cocal and VSV-G pseudotypes. The RD114/TR pseudotypewas relatively resistant to neutralization in dog serum and may bebetter for in vivo delivery in this model. The lower titers obtainedusing RD114/TR limit its utility for in vivo delivery applicationsrequiring transduction of a large numbers of cells. The RD114 pseudotypeis effective for therapeutic delivery in the canine X-SCID setting usingoncoretroviral vectors where corrected cells have a selective advantageand small numbers of transduced cells can expand and cure the diseasephenotype. Ting-De Ravin et al., Blood 107:3091-3097 (2006).

Example 5 Efficient Transduction of Hematopoietic Progenitors with CocalPseudotyped Lentiviral Vectors

Hematopoietic stem cells are an attractive target for gene therapyapplications since these cells can be easily harvested and transduced exvivo then reintroduced to reconstitute the entire hematopoietic system.Lentiviral vectors pseudotyped with VSV-G allow for efficienttransduction of repopulating cells in nonobese-diabetic severe combinedimmunodeficient (NOD/SCID) xenotransplantation assays for humanrepopulating cells (Miyoshi et al., Science 283:682-686 (1999)) and inlarge animal models. Trobridge et al., Blood 111:5537-5543 (2008); Hornet al., Blood 103:3710-3716 (2004); and Hanawa et al., Blood103:4062-4069 (2004). Lentiviral vectors pseudotyped with the RD114/TRenvelope efficiently transduce human and primate hematopoieticprogenitor cells (Sandrin et al., Blood 100:823-832 (2002); Hanawa etal., Mol. Ther. 5:242-251 (2002)), and when directly compared at asimilar MOI are more efficient for gene transfer to human CD34⁺ NOD/SCIDrepopulating cells than VSV-G. Di Nunzio et al., Hum. Gene Ther.18:811-820 (2007).

The efficiency of transduction of CD34⁺ hematopoietic progenitors fromhumans, pigtailed macaques (Macaca nemestrina), and canines werecompared using the three different envelope pseudotypes. Transductionwas detected in CD34-derived cells in liquid culture by flow cytometryand also by visual scoring of EGFP-positive CFUs by fluorescentmicroscopy (FIG. 4). The Cocal envelope allowed for efficienttransduction of hematopoietic progenitors from humans, macaques, anddogs. Analysis of the plating efficiency for all three pseudotypes werecompared in triplicate at the same MOI (5) in human cultures and showedthat there was no significant difference in the relative ability ofhematopoietic progenitors to form colonies after exposure to vectorpreparations (p=0.12). Thus, there was no difference in toxicity tohematopoietic progenitors between vector preparations of the differentpseudotypes. Transduction of pigtailed macaque CD34⁺ cells with theCocal envelope was slightly more efficient than with VSV-G or RD114/TRwhen compared at the same MOI, similar to what was observed in pigtailedmacaque stromal cells and skin fibroblasts. Together, these datademonstrate that Cocal pseudotyped lentiviral vectors efficientlytransduced hematopoietic progenitor cells, suggesting that they may behighly effective for HSC gene therapy.

Example 6 Efficient Transduction of Pigtailed Macaque Repopulating Cellswith Cocal Pseudotyped Lentiviral Vectors

Given the high efficiency of transduction of macaque and humanhematopoietic progenitors using Cocal pseudotyped vectors, the relativeability of Cocal and VSV-G pseudotyped vectors to transduce long-termrepopulating cells was compared in a clinically relevant nonhumanprimate model. A competitive repopulation model was employed to directlycompare transduction and engraftment of VSV-G and Cocal pseudotypedlentiviral vectors in the same animal. In this approach, a lentiviralEGFP vector was pseudotyped with VSV-G, and a second lentiviral vectorthat expresses enhanced yellow fluorescent protein (EYFP) reporter genewas pseudotyped with Cocal envelope. The green and yellow proteinsdiffer only by five amino acids and are thus immunologically highlysimilar. Repopulating cells expressing these fluorophores can be easilydistinguished and accurately quantitated in vivo by flow cytometry.CD34⁺ cells were transduced separately at an MOI of 20 using eachpseudotype, extensively washed, and then sequentially infused into amyeloblated (1020 cGy total body irradiation) macaque. Using thisapproach, hematopoietic repopulating cells could be transduced by eitherthe EGFP-expressing Cocal pseudotyped vector or the EYFP-expressingVSV-G pseudotyped vector, but not both. Analysis of pretransplantmarking showed approximately 4.5-fold higher marking in the Cocal arm inliquid cultures at day 11 post-vector exposure and marking in CFUs wassimilar to these results in liquid cultures (Table 2).

TABLE 2 Pre-infusion Transduction Efficiency and Engraftment ofPigtailed Macaque CD34+ Cells No. of CD34− enriched cells/ No. of Dayskg × 10⁶ Envelope Infused Pre-infusion to Days to CD34+ beforeGlyoprotein/ Cells/ Transduction ANC Platelets Monkey* Purity cultureFluorophore kg × 10⁶ MOI** Efficiency*** >500 >20,000 L06348 99% 7.1Cocal/EGFP 41 20  17%, 17% 14 25 (2.8 kg) 7.1 VSV- 32 20 3.8%, 5% G/EGFP *Monkey and weight at time of transplantation in brackets**Multiplicity of infection based on titer determined by transduction ofHT1080 cells ***Percentage of fluorescence-positibe cells assessed yflow cytometry for EGFP or EYFP in liquid cultures on day 11 aftertransduction, and percentage of fluorescence-positive colony formingunits

Engraftment was rapid with 14 days to an ANC greater than 500/μl and 25days to a platelet count greater than 50,000/μl. Transduced cells in theperipheral blood were then quantitated in vivo by flow cytometry (FIG.5). The data demonstrated that the Cocal pseudotyped vector allowed forhighly efficient gene transfer with up to 20% marking as assessed bytransgene expression in granulocytes and 6% in lymphocytes. Marking inthis animal as assessed by transgene expression was approximately2.8-fold higher for the Cocal arm than for the VSV-G arm in granulocytesand 2.2-fold higher in lymphocytes.

At a relatively low MOI (5), lentiviral vectors pseudotyped with Cocalenvelope consistently transduce pigtailed macaque cells more efficientlythan VSV-G pseudotyped vectors while exhibiting similar transductionefficiency in human cells. The transduction efficiency in pigtailedmacaque cells closely reflects the efficiency in human cells, suggestingthat Cocal envelope may be a better pseudotype than VSV-G for genetransfer studies in the pigtailed macaque model, since it may be morepredictive of gene transfer in patients.

Nonhuman primates are particularly useful for HSC gene therapy sinceprimates very closely model hematopoiesis in humans. The pigtailedmacaque in particular is an excellent model to evaluate HIV-basedlentiviral vector transduction because, unlike rhesus macaques,pigtailed macaque repopulating cells can be efficiently transduced usingVSV-G pseudotyped HIV-based vectors that also efficiently transducehuman cells. Trobridge et al., Blood 111:5537-5543 (2008). Transductionof HSCs is limited by several factors which include the quiescence oftarget long-term repopulating cells, but also the availability of thehost envelope receptor that mediates efficient entry of viral vectors.Sabatino et al., Blood Cells, Molecules, and Diseases 23:422-433 (1997)and Kurre et al., J. Virol. 73:495-500 (1999).

As part of the present disclosure, higher marking in macaquerepopulating cells using the Cocal pseudotyped vectors was observed upto 113 days post-transplant. Marking will be monitored longer term (>1year) to determine if there are any obvious differences between VSV-Gand Cocal envelope in lineage marking of longer-term repopulating cells.Cocal envelope can mediate efficient multi-lineage gene transfer tomacaque long-term (>90 days) repopulating cells. Additional studies areperformed to confirm that Cocal pseudotyped vectors are more efficientthan VSV-G for HSC gene transfer in this model.

Example 7 Pseudotyping Gammaretroviral Vectors

A Moloney leukemia virus-based vector that expresses EGFP was producedby transient transfection with cocal, VSV, and RD114/TR envelopeglycoproteins, the vector supernatants were concentrated bycentrifugation and the titers were compared (FIG. 11). Cocal was foundto efficiently pseudotype gammaretroviral vectors and to allowconcentration of these vectors. These data extends the use of cocalenvelope for use with gammaretroviral vectors, and suggests it may be aversatile envelope for additional enveloped vector types.

Example 8 Production of a Human Embryonic Kidney 293C Packaging Cellthat Constitutively Produces Cocal Envelope

One limitation of the VSV-G envelope is that it is difficult to producehigh-titer packaging cells that constitutively express VSV-G. Togenerate a packaging cell that would constitutively produce cocalenvelope, human embryonic kidney 293 cells were transfected with thecocal envelope plasmid and with a plasmid that expresses a blasticidinresistance gene. Blasticidin-resistant colonies were isolated and eachindividual cell line was screened for their ability to stably producecocal envelope using an EGFP-expressing vector. A packaging cell wasidentified (named 293C for 293 Cocal) that constitutively produces cocalenvelope as evidenced by the ability to produce high titer lentiviralvector when transfected with vector and helper plasmids in the absenceof an envelope plasmid. This 293C cell line produced high titerlentiviral vector (FIG. 12) that can be efficiently concentrated bycentrifugation. 293C shows vector made from the 293C cell line, the‘Cocal transient’ and ‘VSV-G transient’ controls show vector made fromtransiently co-transfecting cocal and VSV-G envelope plasmids into 293cells. This cell line was generated from a single colony and culturedand expanded for over 6 weeks, so expression of the cocal envelope isstable. This 293C packaging cell line may have broad utility for theproduction of many types of viral vectors including vectors for genetherapy and for vaccines. Vector producing cells may be generated usingthis 293C packaging line.

Example 9 Production of a Phoenix-Cocal Packaging Cell thatConstitutively Produces Cocal Envelope and Also Gammaretroviral Gag andPol

Phoenix packaging cells developed by Garry Nolan's research groupexpress gammaretroviral Gag and Pol and produce vector virions whentransfected with a gammaretroviral vector and envelope plasmids. Phoenixpackaging cells were transfected with the cocal envelope plasmid and ablasticidin-resistance plasmid. Stable blasticidin-resistantPhoenix-derived colonies that expressed cocal and produce vector werescreened for their ability to produce cocal-pseudotyped EFGP-expressingvector. One cell line (named Phoenix-C for Phoenix-Cocal) was identifiedthat produced high titer gammaretroviral vector when transfected with avector plasmid only. This cell line was generated from a single colonyand cultured and expanded for over 6 weeks to ensure stable expressionof the cocal envelope. For this Phoenix-C packaging line, concentratedgammaretroviral vector stocks of over 10⁶ EGFP transducing units/ml weregenerated.

Example 10 Transduction of Long Term (>1 Year) Repopulating Cells in aNonhuman Primate Model

Cocal-pseudotyped vectors have been shown to be capable of efficientlytransducing pigtailed macaque (Macaca nemestrina) hematopoieticrepopulating cells. Marking to over 1 year has been demonstrated in thisanimal. Marking was compared using a competitive repopulation assay withEGFP (cocal) and EYFP (VSV-G) experimental arms. After 1 year, markingin the cocal arm remained higher than the VSV-G arm in both myeloid andlymphoid cells. These data showed that cocal envelope could mediateefficient gene transfer to long term repopulating cells in a clinicallyrelevant large animal model. See FIG. 13

Example 11 Generation of a Gammaretroviral Producer Cell Line Based onthe Phoenix-Cocal Packaging Cell

The Phoenix-cocal packaging cell was transfected with a gammaretroviralvector plasmid (pMND-EGFP-SN-/-) and a puromycin resistance plasmidpPur. Puromycin-resistant colonies were screened for production ofEGFP-expressing vector. A stable producer line was obtained that stablyproduces the gammaretroviral vector after over 5 weeks in culture. Thevector from this stable producer cell line was concentrated 100-fold bycentrifugation to a titer of 7.2×10⁷ EGFP transducing units/ml.High-titer gammaretroviral producer cell lines were generated thatstably expressed cocal envelope allowing for the production of vectorsin the cell supernatant that can be efficiently concentrated to hightiter. This also suggests that stable cocal-pseudotyped producer celllines can be generated for other vector types including lentiviralvectors. Cocal pseudotyped producer cells can be used for manyscientific and therapeutic applications including gene therapy and theproduction of reprogramming vectors to generate induced pluripotent stemcells for broad application in regenerative medicine.

1.-28. (canceled)
 29. A method for delivering a gene of interest to atarget cell, said method comprising the step of contacting said targetcell with a Cocal vesiculovirus envelope pseudotyped retroviral vectorparticle.
 30. The method of claim 29 wherein said Cocal vesiculovirusenvelope pseudotyped retroviral vector particle is selected from thegroup consisting of a lentiviral vector particle, an alpharetroviralvector particle, a betaretroviral vector particle, a gammaretroviralvector particle, a deltaretroviral vector particle, and anepsilonretroviral vector particle.
 31. The method of claim 29 whereinsaid Cocal vesiculovirus envelope pseudotyped retroviral vector particlecomprises a Cocal vesiculovirus envelope protein encoded by thenucleotide sequence of SEQ ID NO:
 1. 32. The method of claim 29 whereinsaid Cocal vesiculovirus envelope pseudotyped retroviral vector particlecomprises a Cocal vesiculovirus envelope protein comprising the aminoacid sequence of SEQ ID NO:
 2. 33. The method of claim 29 wherein saidCocal vesiculovirus envelope pseudotyped retroviral vector particlecomprises a Cocal vesiculovirus envelope protein encoded by thenucleotide sequence of SEQ ID NO:
 3. 34. The method of claim 29 whereinsaid Cocal vesiculovirus envelope pseudotyped retroviral vector particlecomprises a Cocal vesiculovirus envelope protein comprising the aminoacid sequence of SEQ ID NO:
 4. 35. A method for delivering a gene ofinterest into a hematopoietic system of a patient, said methodcomprising the steps of: (a) harvesting a CD34⁺ hematopoietic stem cell(HSC) from said patient; (b) transducing said HSC ex vivo with a Cocalvesiculovirus envelope pseudotyped retroviral vector particle comprisingsaid gene of interest; and (c) introducing said ex vivo transduced HSCinto said patient under conditions that allow reconstitution of saidpatient's hematopoietic system.
 36. A method for identifying a patientsusceptible to treatment with a Cocal vesiculovirus envelope pseudotypedretroviral vector particle; said method comprising the steps of: (a)isolating a serum sample from said patient; (b) contacting said Cocalvesiculovirus envelope pseudotyped retroviral vector particle with saidserum sample; and (c) testing said Cocal vesiculovirus envelopepseudotyped retroviral vector particle for serum inactivation.